Stroke is the third leading killer in the US and the main cause for over 795,000 cases of adult disability each year. The two major types of stroke, ischemic and hemorrhagic, cannot be clinically differentiated; 30% of patients presenting stroke-like symptoms do not have a stroke at all. It is imperative that a stroke diagnosis be made quickly and accurately because ischemic and hemorrhagic strokes require different treatments that have only a small window of time to be effective (3-6 hours). Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used for the diagnosis, which delays therapeutic intervention. Unfortunately, a molecular diagnostic test does not currently exist for stroke due primarily to the fact that potential molecular marker panels are waiting to be clinically validated. This validation process is, in part, hampered by the tools used for their analysis, such as reverse-transcription PCR (RT-PCR) for nucleic acid-based markers or immunoassays for serum protein markers, due to the lack of sample process automation and the lengthy processing steps used. In this application, a highly innovative, fully-automated system will be developed that has exquisite analytical sensitivity to monitor minute changes in the expression levels of different molecular markers with a turn-around-time (TAT) of less than 15 minutes. The system will facilitate the identification and validation of new molecular markers for the rapid, specific, and sensitive diagnosis of stroke. The molecular assay and novel hardware will be used to evaluate the potential of a messenger RNA marker panel as an unprecedented blood-based test (using peripheral blood mononucleated cells, PBMC) for the diagnosis of stroke. Two technologies will form the core of the system: (1) modular fluidic bio-processor made from polymers via replication that contains all of the sample processing steps; and (2) single pair fluorescence resonance energy transfer (spFRET) that eliminates several sample processing steps. Micro-replication will be used for the fabrication of the bio-processor to keep cost low and make this consumable appropriate for single- use applications, as demanded by clinical diagnostics. The bio-processor will contain a fluidic motherboard with task-specific modules interconnected on it to provide design flexibility. The molecular processing steps poised on the processor will be: (1) PBMC isolation; (2) PBMC lysis; (3) mRNA solid-phase purification; (4) reverse transcription to cDNA; (5) ligase detection reactions (LDRs) to generate molecular beacons with donor/acceptor fluorescent pairs and; (6) spFRET digital detection. The molecular (i.e., digital) counting using spFRET will provide the ability to eliminate several sample pre-processing steps shortening the assay TAT and generate the necessary analytical sensitivity to detect subtle changes in expression levels of mRNA markers, which may be critical during early times following a stroke event. The system can potentially be used for any application requiring mRNA expression profiling, but will be used in the current project for validating molecular markers for the diagnosis of stroke. PUBLIC HEALTH RELEVANCE: Stroke is the third leading killer in the US and causes over 795,000 cases of adult disability each year. Unfortunately, in vitro diagnostic tests for stroke that can be administered to patients for effective and timely treatment are not currently available. The goal of this project is to develop an innovative, discovery-based technology for securing highly quantitative results quickly through full-process automation. The technology will assist in the validation of different molecular markers for stroke to deliver an unprecedented in vitro diagnostic blood-based test.